PROPOSAL SUMMARY Live attenuated vaccines have proven to be the most efficient human vaccines for many serious infectious diseases. When compared to their dead counterparts, live vaccines induce superior immune protection and lasting memory. But despite the efficacy of live vaccines, concerns over their safety have led to vaccine refusal by some and withholding their administration to the very young, the elderly and immunocompromised. Preservation and delivery of live vaccines especially to impoverished areas in developing countries is difficult and expensive. Understanding the molecular basis for the efficacy of live vaccines is significant because it would enable targeting of the relevant immune pathways that induce optimal and long-lasting protective immunity. Importantly, it would set the stage for the development of vaccines that are safe and afford the same protection as live vaccines, alleviating public fears and increasing the segment of the population that is vaccinated. We began our work eight years ago with the hypothesis that innate immune cells sense microbial viability as a distinct set of pathogen associated molecular patterns (PAMPs), and we identified bacterial messenger RNA (mRNA) as a vita-PAMP that signifies bacterial viability and mobilizes a tailored immune response not warranted for dead microorganisms. The Toll-like receptor (TLR) signaling adaptor TRIF plays a central role here upstream of inflammatory type I interferon and NLRP3 inflammasome pathways. Adding bacterial mRNA to dead bacteria recapitulates these innate responses, and supplementing a dead vaccine with bacterial mRNA (what we call a vita-vaccine) augments its performance in mice. A vita-vaccine performed similarly to a live vaccine in uniquely eliciting a follicular T helper cell response (that helps B cells), germinal center formation, and B cell isotype class switching, all in a TRIF-dependent manner. These studies provide strong evidence that vita-vaccine versions of existing vaccines could represent a significant advance in being able to combine the efficacy of live vaccines with the safety of dead vaccines. The three overlapping areas we will investigate in this project are: 1. We will determine how adaptive immunity elicited by the supplementation of a dead bacterial vaccine with the vita-PAMP bacterial mRNA compares to that elicited by PAMPs such as bacterial lipopeptides and others. 2. We will investigate how bacterial mRNA impacts the performance of subunit vaccines. We will test vita- vaccine versions of the licensed anthrax subunit vaccine and Influenza A virus monovalent subunit vaccine. 3. We will test a vita-vaccine version of a trivalent inactivated Influenza virus vaccine and compare it to the live attenuated influenza vaccine. The completion of these studies should provide sufficient experimental evidence to warrant the use of bacterial mRNAs as superior vita-adjuvants that restore the signatures of microbial viability to dead vaccines and improve existing inactivated and subunit vaccines for protection against either bacterial or viral diseases.